Strains of the genus Corynebacterium, in particular the species Corynebacterium glutamicum, are known producers of L-amino acids, such as proteinogenic amino acids, e.g. L-lysine, L-threonine, L-valine or L-isoleucine, and of other fine chemicals, such as vitamins, nucleosides and nucleotides. Because of the great economic importance of these chemicals work is continually being done on improving the production methods. Improvements may relate to the genetic constitution of the microorganism, to the fermentation technology applied or to the working-up to the desired product form. The methods used for improving the genetic constitution are those of mutagenesis, selection and choice of mutants. Methods of recombinant DNA technology have likewise been used for a number of years for strain improvement of this group of bacteria. Background summaries concerning Corynebacterium, in particular Corynebacterium glutamicum, may be found in L. Eggeling and M. Bott (Handbook of Corynebacterium glutamicum, CRC Press, 2005), A. Burkovski (Corynebacteria Genomics and Molecular Biology, Caister Academic Press, 2008) or H. Yukawa and M. Inui (Corynebacterium glutamicum Biology and Biotechnology, Springer Verlag, 2013).
One of the main carbon sources used for propagation of this group of bacteria and for formation of a desired chemical is glucose. Glucose used in the fermentation industry is typically produced from starch by enzymatic hydrolysis. Starch is a mixture of two different polysaccharides each consisting of chains of linked, repeated glucose units. The mixture mainly consists of two separate polysaccharides, amylose and amylopectin. Amylose is an almost linear polysaccharide with glucose units connected almost exclusively through α-1,4 glycosidic linkages. Glucose units in amylopectin are additionally linked through α-1,6 glycosidic linkages. The content of amylose in starch in plant species like maize, wheat or rice is about 20 to 30% and the amylopectin content about 80 to 70%. Detailed information about starch may be found in J. Bemiller and R. Whistler (Starch: Chemistry and Technology, 3. ed., Elsevier, 2009).
Enzymatic starch hydrolysis to glucose involves two main steps. In the first step, also referred to as liquefaction, starch is treated with α-amylase (4-α-D-glucan glucanohydrolase; EC 3.2.1.1). The products of this reaction are α-1,4 linked oligomers of glucose, also referred to as maltodextrin, comprising molecules like maltotriose (O-α-D-Glcp-(1→4)-O-α-D-Glcp-(1→4)-D-Glcp) and maltohexaose (the respective α-(1→4)-linked D-glucose hexamer), and oligomers of glucose containing an α-1,6 linkage also referred to as limit dextrin. In the second step, also referred to as saccharification, this mixture is treated with glucoamylase, also referred to as amyloglucosidase in the art (4-α-D-glucan glucohydrolase; EC 3.2.1.3). This enzyme hydrolyzes the α-1,4 linkage rapidly. It also hydrolyzes the α-1,6 linkage but at a slower rate. The art also describes the use of pullulanase (pullulan 6-α-glucanohydrolase) in order to hydrolyze the α-1,6 linkage contained in the limit dextrins. The product of this second step is a glucose solution containing amongst others residual maltose (4-O-(α-D-Glucopyranosyl)-D-Glucopyranose), isomaltose (6-O-(α-D-Glucopyranosyl)-D-Glucopyranose) and panose (O-α-D-Glcp-(1→6)-O-α-D-Glcp-(1→4)-D-Glcp) as side products. These side products are the result of reverse enzymatic reactions due to the high glucose concentration accumulating during the saccharification step. The reverse reaction of glucoamylase yields maltose and isomaltose. As commercial enzyme preparations may contain transglucosidase (1,4-α-glucan 6-α-glucosyltransferase; EC 2.4.1.24) the presence of this enzyme also contributes to the formation of isomaltose and panose. Many variations of this basic procedure exist due to the enzymes available, mixtures thereof and reaction conditions.
Summaries concerning the enzymatic hydrolysis of starch to glucose and the side products formed may be found in P. H. Blanchard (Technology of Corn Wet Milling and Associated Processes, Elsevier, 1992), M. W. Kearsley and S. Z. Dziedzic: Handbook of Starch Hydrolysis Products and their Derivatives, Chapmann & Hall, 1995), B. H. Lee (Fundamentals of Food Biotechnology, VCH Publishers, 1996) or H. Uhlig (Industrial Enzymes and their Application, John Wiley & Sons 1998). Data concerning the composition of starch hydrolysates thus manufactured may be found in A. Converti (starch/starke 46 (7), 260-265, 1994), M. Chaplin and C. Bucke (Enzyme Technolgy, Cambridge University Press, 1990), Amarakone, P. et al (Journal of the Japanese Society of Starch Science, 31(1), 1-7, 1984), WO9927124 A1 and WO2005100583 A2. The glucose content of such starch hydrolysates is approximately 85 to 97% (based on dry matter content).
For industrial fermentative production of commodity fine chemicals like L-amino acids, e.g. L-lysine, it is not economical to first purify glucose from starch hydrolysate and then use it in the fermentation process. Instead starch hydrolysate itself is used as a low cost, glucose containing feedstock.
Corynebacterium glutamicum is unable to use isomaltose or panose as a carbon source. Accordingly these compounds accumulate in the fermentation broth during a production process when said starch hydrolysate is used as feedstock. The presence of these sugars in turn is unfavourable because they are an additional load to the plants waste water. Further they may generate product losses during the processing steps for manufacturing the final product. For example it is known that the reducing end of a sugar molecule may react with the amino group of L-amino acids, e.g. L-lysine, to give Maillard reaction products (M. W. Kearsley and S. Z. Dziedzic: Handbook of Starch Hydrolysis Products and their Derivatives, Chapmann & Hall, 1995).
In order to avoid these disadvantages methods were developed to convert isomaltose and/or panose to glucose during a fermentation process. WO2005100583 A2, WO2014093312 A1 and WO2015061289 A1 describe the addition of transglucosidase to the fermentation broth containing starch hydrolysate or sugar syrup as carbon source. This approach has the disadvantage that the enzyme must be produced separately thus adding to the production costs.
A different approach was followed by EP2241632 A1. It is suggested to impart a microorganism with an isomaltase activity. As microorganisms Enterobacteriaceae including E. coli and coryneform bacteria, including specific examples of this group of bacteria are presented. EP2241632 A1 further teaches that an intracellular or an extracellular isomaltase can be used. In case an intracellular isomaltase is imparted and the cell does not possess an activity to take up isomaltose it is preferred to impart both the intracellular isomaltase activity and the activity to take up isomaltose into the cell. As examples for an isomaltase gene the genes malL and glvA of Bacillus subtilis and homologues thereof are shown. As isomaltose transporter genes the glvC gene of Bacillus subtilis and other genes fulfilling a similar function of various origin are shown. During examination proceedings an experimental example was presented in which the glvA and the glvC gene of Bacillus subtilis were expressed in an L-lysine excreting strain of C. glutamicum. The strain constructed showed favorable isomaltose consumption and L-lysine formation as compared to the reference. However, EP2241632 A1 is silent whether this system will enable a C. glutamicum cell to consume panose.
EP2241632 A1 further generally proposes that an extracellular isomaltase gene may be obtained by ligating the coding region of the isomaltase gene with a sequence coding for a signal peptide for secreting the protein into a cell surface layer or out of the cell. As signal peptide the protein A of Staphylococcus aureus is suggested. A technical example is given for E. coli by fusing said signal peptide of protein A to the MalL isomaltase of Bacillus subtilis. The document is silent whether this secreted isomaltase also attacks panose. Furthermore, the document is silent about suitable signal peptides for Corynebacterium glutamicum or how to elect an appropriate signal peptide fitting to the isomaltase.
EP2241632 A1 also presents two lists of putative isomaltase genes from various microrganisms. Table 1 of EP2241632 A1 presents potential isomaltases as homologues of MalL having the function of amongst others oligosaccharide-producing multifunctional G-amylase, oligo-1,6-glucosidase, alpha amylase catalytic region or trehalose-6-phosphate hydrolase. Table 2 presents potential isomaltase genes as homologues having the function of maltose-6′-phosphate glucosidase or 6-phospho-alpha glucosidase.
Similarly, S. Jiang and L. Ma disclosed the nucleotide sequence of an oligo-1,6-glucosidase gene of Bacillus subtilis strain HB002 (available at the National Center for Biotechnology Information (NCBI) under GenBank accession number AY008307.1). The entry is silent about the activity of the encoded protein towards isomaltose and panose.
The art teaches various intracellular α-1,6-glucosidases (EC 3.2.1.10) having the ability to attack the α-1,6 linkage of isomaltose and/or panose.
The nucleotide sequence of the IMA1 gene of Saccharomyces cerevisiae strain S288c encoding an oligo-1,6-glucosidase is available at the NCBI under GenBank accession number NC_001139 having the locus_tag YGR287C. The entry discloses the encoded protein as an isomaltase. The entry is silent about its activity towards panose.
The dexB gene of Streptococcus mutans encodes an intracellular glucan 1,6-alpha-glucosidase (Whiting et al, Journal of General Microbiology 139, 2019-2026, 1993) having the ability to hydrolize the α-1,6 linkage in isomaltose and panose.
WO2004018645 A2 relates to the sequencing of the genome of Bifidobacterium breve ATCC 15700 and in particular to the identification of genes encoding enzymes having the ability to hydrolyze the α-1,6 linkage in isomaltose and panose.
Pokusaeva et al (Applied and Environmental Microbiology 75, 1135-1143, 2009) describe two genes agl1 and agl2 of Bifidobacterium breve UCC2003 encoding the enzymes Agl1 and Agl2, both having the activity of α-1,6-glucosidases. The enzymes were able to hydrolyze the α-1,6 linkage in panose and isomaltose. Pokusaeva et al. make no explicit statement about the intra- or extracellular location of the two enzymes. However in a review article by Pokusaeva et al. (Genes and Nutrition 6, 285-306, 2011) the two enzymes Agl1 and Agl2 are classified as “cytoplasmic enzymes” (see page 299-300).
In C. glutamicum two pathways for the secretion of proteins exist. One is referred to as Sec-pathway and mediates translocation of preproteins in an unfolded state through the membrane. The other is referred to as Tat-pathway and mediates transfer of preproteins in their folded state. The signal peptide of the preprotein is cleaved of from the preprotein by a peptidase during the secretion process and the mature protein is released into the culture medium. Summaries concerning protein secretion in Corynebacterium glutamicum were presented by A. A. Vertes contained in H. Yukawa and M. Inui (Corynebacterium glutamicum Biology and Biotechnolgy, Springer Verlag, 2013) and Liu et al (Critical Reviews in Biotechnology 1-11, 2016).
There are a number of reports for successful secretion of different proteins from different species or origin in C. glutamicum. However most of these proteins are secreted by their natural hosts indicating that these proteins have an intrinsic ability of being secretable.
Liebl et al. (Journal of Bacteriology 174, 1854-1861, 1992) reported on the successful expression and secretion of a staphylococcal nuclease, an extracellular enzyme of Staphylococcus aureus in C. glutamicum using the signal peptide of the original host.
Billman-Jacobe et al. (Applied and Environmental Microbiology 61, 1610-1613, 1995) report on expression and secretion of the basic protease of Dichelobacter nodosus and the subtilisin of Bacillus subtilis in C. glutamicum. While the secretion of subtilisin was directed by its own signal peptide the natural signal peptide of the basic protease did not facilitate secretion. After replacement of the natural signal sequence by the subtilisin signal sequence the basic protease was secreted by C. glutamicum. 
Salim et al. (Applied and Environmental Microbiology 63, 4392-4400, 1997) report on the expression and secretion of antigen 85 protein of Mycobacterium tuberculosis in C. glutamicum. This protein is naturally found in the culture filtrates of M. tuberculosis. 
EP1375664 A1 relates to the production and secretion of heterologous proteins such as the pro-transglutaminase of Streptoverticillium mobarense or the human epidermal growth factor (hEGF) in Corynebacterium glutamicum by fusing said proteins with signal peptide sequences of cell surface proteins of C. glutamicum or C. ammoniagenes. The pro-transglutaminase of Streptoverticillum mobarense is an enzyme which is secreted by its natural host (Pasternack et al; European Journal of Biochemistry 257, 570-576, 1998). The human epidermal growth factor is a secreted peptide originally found in human urine by Cohen, S. and Carpenter, G. (Proceedings of National Academy of Sciences USA 72(4), 1317-1321, 1975).
EP1748077 A1 relates to the production and secretion of heterologous proteins in coryneform bacteria making use of a Tat system-dependent signal peptide region. In particular isomalto-dextranase of Arthrobacter globiformis (a 6-α-D-glucan isomaltohydrolase) was secreted by C. glutamicum using the signal sequence of the isomalto-dextranase or the signal sequence of the cell surface layer protein SIpA of C. ammoniagenes. Protein glutaminase of Chryseobacterium proteolyticum was secreted by C. glutamicum using the isomaltodextranase signal sequence of A. globiformis, the SIpA signal sequence of C. ammoniagenes or the TorA signal sequence of Escherichia coli. The isomalto-dextranase of Arthrobacter globiformis is an enzyme which is secreted by its natural host (Iwai et al; Journal of Bacteriology 176, 7730-7734, 1994). The protein glutaminase of Chryseobacterium proteolyticum is also an enzyme which is secreted into the culture medium by its natural host (Kikuchi et al; Applied Microbiology and Biotechnology 78, 67-74, 2008).
Watanabe et al. (Microbiology 155, 741-750, 2009) identified the N-terminus of the CgR0949 gene product and other gene products of C. glutamicum R as signal peptides addressing the Tat secretory pathway for proteins. The CgR0949 signal sequences comprises a sequence of 30 amino acid residues. After addition of this signal amino acid sequence to the α-amylase of Geobacillus stearothermophilus from which the natural signal peptide was removed the enzyme was secreted by the C. glutamicum host into the culture medium. The α-amylase of Geobacillus stearothermophilus is an enzyme which is secreted by its natural host (Fincan and Enez, Starch 66, 182-189, 2014).
Breitinger, K. J. (Dissertation/Ph.D. Thesis Ulm University 2013) disclosed the expression of a fusion polypeptide composed of the putative signal sequence of the protein encoded by gene cg0955 of C. glutamicum ATCC 13032 and the pullulanase PulA of Klebsiella pneumoniae UNF5023 in an L-lysine producing strain of C. glutamicum. Pullulanase activity was detected in the cell lysate and in the membrane fraction of said C. glutamicum cells but not in the culture supernatant of said strain. The pullulanase PulA of Klebsiella pneumoniae UNF5023 is an enzyme which is secreted by its natural host (Kornacker and Pugsley, Molecular Microbiology 4, 73-85, 1990). Breitinger, K. J. further stated that the 5′-terminus of gene Cg0955 of C. glutamicum ATCC 13032 shows a 95% homology to the signal sequence of gene cgR0949 of C. glutamicum R. The signal sequence of the protein encoded by gene cgR0949 was classified as a Tat-type signal sequence by Watanabe et al. (Microbiology 155, 741-750, 2009).
Hyeon et al (Enzyme and Microbial Technology 48, 371-377, 2011) constructed vector pMT1s designed for secretion of gene products into the culture medium using the cg0955 nucleotide sequence encoding the Tat signal peptide. Thus they were able to achieve secretion of the CbpA scaffolding protein of Cellulomonas celluvorans and the endoglucanase CelE of Clostridium thermocellum in C. glutamicum to form minicellulosomes. These proteins are secreted and displayed on the cell surface in their natural hosts.
Kim et al (Enzyme and Microbial Technology 66, 67-73, 2014) similarly expressed and secreted the endoglucanase CelE and the β-glucosidase BglA of C. thermocellum in C. glutamicum to display them on the cell surface. In their natural host these enzymes are constituents of cellulosomes located on the cell surface of its host.
Matano et al (BMC Microbiology 16, 177, 2016) studied the expression and secretion of N-acetylglucosaminidase from different microorganisms. A gene termed nagA2 was identified in the chromosome of C. glutamicum. After its expression enzyme activity was detected in the cytoplasmic fraction and culture supernatant. After replacement of the putative signal peptide of NagA2 with different Tat-type signal sequences including SP0955 (another term for the signal peptide encoded by cg0955) secretion efficiency was improved.
Matano et al. further achieved secretion of the exochitinase ChiB of Serratia marcescens by fusing the sequence encoding the Tat secretion signal peptide from the C. glutamicum gene cg0955 to chiB. It is noted that the exochitinase ChiB of Serratia marcescens is an enzyme which is exported into the periplasm by its natural host (Brurberg et al, Microbiology 142, 1581-1589 (1996)). Matano et al. further investigated the secretion of the Bacillus subtilis N-acetylglucosaminidase encoded by nagZ in C. glutamicum. This enzyme is inefficiently secreted by its natural host. NagZ N-acetylglucosaminidase was also expressed with various C. glutamicum signal peptides to increase the amount of enzyme in the supernatant. However, fusion to these signal peptides including the signal peptide from Cg0955 had no effect on the amount of enzyme secreted into the culture supernatant. In particular it is noted that fusion to the signal peptide from Cg0955 drastically increased the amount of intracellular enzyme activity.
Yim et al. (Applied Microbiology and Biotechnology 98, 273-284, 2014) report on the secretion of a recombinant single-chain variable antibody fragment against anthrax toxin in C. glutamicum. The use of the TorA signal peptide addressing the Tat pathway resulted in negligible secretion whereas the use of the PorB signal peptide addressing the Sec pathway resulted in measurable secretion. The authors also stated that the use of a codon optimized gene sequence was one of the components for high production of the protein.
WO2008049782 A1 relates to increasing gene expression in C. glutamicum by adjusting the codon usage of genes to that of abundant proteins in the host cell.
The green fluorescent protein (GFP) has attracted much interest in molecular biology as a model protein easy to monitor due to its fluorescence. It is found in jellyfish like Aequora victoria, where it is localized in specialized photocytes (J. M. Kendall and M. N. Badminton, Tibtech, 216-224, 1998). Meissner et al. (Applied Microbiology and Biotechnology 76, 633-642, 2007) investigated protein secretion using the green fluorescent protein in three different Gram-positive bacteria Staphylococcus carnosus, Bacillus subtilis and Corynebacterium glutamicum. In all three microorganisms fusion of a Tat-signal peptide to GFP resulted in its translocation through the cytoplasmic membrane. However, in S. carnosus GFP was trapped entirely in the cell wall and not released into the supernatant. In Bacillus subtilis GFP was secreted into the supernatant in an inactive form. In C. glutamicum different Tat signal peptides were used. The TorA signal peptide from E. coli, the PhoD signal sequence of C. glutamicum and the PhoD signal sequence of Bacillus subtilis. Although GFP was secreted in all three cases the amount of secreted protein was significantly different. Strikingly the PhoD signal sequence from B. subtilis gave the best result.
Teramoto et al. (Applied Microbiology and Biotechnoogy 91, 677-687, 2011) used the signal peptide of CgR0949 to achieve high yield secretion of GFP in C. glutamicum. 
It is noted that Hemmerich et al. (Microbial Cell Factory 15(1), 208, 2016) after a search for a suitable signal peptide for the secretion of the cutinase of Fusarium solani pisi in Corynebacterium glutamicum concluded that the best signal peptide for a specific target protein has to be evaluated each time from scratch.
Isomaltose and/or panose are contained in starch hydrolysate in comparably small amounts. Accordingly, for a research program aiming at a C. glutamicum strain producing a fine chemical, e. g. L-lysine, at high yield and using the comparatively low amounts of these sugars as additional carbon source, it is not desirable to produce and secrete an enzyme, hydrolyzing the α-1,6 glycosidic linkage of these sugars, at high yield. Both compounds, the fine chemical and the enzyme, would compete for the same carbon source(s) and thus the yield of the compound of commercial interest, which is the fine chemical, would be negatively affected. The enzyme produced and secreted would then be a metabolic burden for the producer of the fine chemical.
Hitherto, directing an intracellular enzyme of a microorganism having the ability to hydrolyse the α-1,6 glycosidic linkage of isomaltose and/or panose, to the extracellular matrix, i. e. the culture supernatant, has not been demonstrated for Corynebacterium glutamicum. 
However, it is desirable to provide a fermentative process for a fine chemical based on a low cost fermentation raw material containing panose and/or isomaltose such as starch hydrolysate using a Corynebacterium, in particular a Corynebacterium glutamicum, having the ability to hydrolyse the α-1,6 glycosidic linkage of panose and/or isomaltose thus making available these glucose oligomers for propagation and fine chemical formation.